Streptococcus mutans has been recognised for many years as the major organism responsible for the development of dental caries in mammals. Various vaccines have been proposed in the past based on various antigenic fragments of S. mutans. One such vaccine is described in British Patent No. 2,060,647 based upon the antigen known as I or I/II. Antigen I has a molecular weight, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of 146-155 Kd. Antigen I/II is believed to be a conjugate of antigen I and antigen II, this I/II antigen having a molecular weight determined by SDS-PAGE of 175-195 Kd. Published European Patent Application No. EP-A-0 116 472 describes antigen X which is a much smaller molecule having a molecular weight, determined by SDS-PAGE of about 3.5-4.5 Kd but which appears to include the same antigenic determinants included within antigens I and I/II.
Antibodies against antigens I, I/II and X are known. The above-mentioned British Patent describes the raising of antibodies against antigens I and I/II by conventional procedures in experimental animals, for example rhesus monkeys, rabbits and mice. These antibodies are proposed primarily for the purification of the antigen by affinity chromatography but the Patent Specification mentions the possibility of using such antibodies for passive immunisation by conventional means. Conventional passive immunisation involves parenteral administration of the antibodies but while such techniques are theoretically available, as a practical matter, passive immunisation has never been regarded as clinically attractive and indeed, the British Patent refers to the preferred use of the antigenic materials for direct immunisation.
All antibodies that have been raised against S. mutans serotype c or against the streptococcal antigen serotype c (SA.sub.c) exhibit a certain degree of cross-reactivity. It is well known that such antibodies are also cross-reactive with antigenic material originating from serotypes e and f of S. mutans. In clinical practice, it is found that serotypes c, e and f amount to about 90% of the bacterial S. mutans population so that the prophylactic or therapeutic use of antibodies raised against serotype c are of considerable practical value but fail to be effective in relation to the residual approximately 10% of the bacterial population. In some series serotype d is found in addition to serotype c in up to 50% of children examined.
This residual 10% is comprised predominantly of a serotype d that until very recently has been regarded merely as another serotype of S. mutans. However recently, this particular serotype has been reclassified as S. sobrinus and, in the description of this invention, we will use the nomenclature S. sobrinus serotype d rather than S. mutans serotype d.
We have now found that if antibodies are raised against S. sobrinus serotype d, that many of the resulting antibodies are cross-reactive not only with S. sobrinus serotype d and also S. sobrinus (S. mutans) serotype g (and serotype a) but surprisingly, that such antibodies are also cross-reactive with serotypes c, e and f of S. mutans. Since somewhere of the order of 98% of all serotypes of S. mutans/S. sobrinus found in the oral cavities are of serotypes a, c, d, e, f and g, our discovery has enabled us to produce, for the first time, antibodies having the potential for prophylactic and/or therapeutic use in relation to substantially 100% of oral bacteria of S. mutans/S. sobrinus type that are responsible for dental caries.
Accordingly, the present invention provides antibodies against a common determinant of S. sobrinus serotypes d and S. mutans serotype c.
The antibodies of the present invention extend not only to whole antibodies but also to antibody fragments containing the necessary binding sites to enable them to recognise and bind with the streptococcal antigen and the antibodies or fragments thereof may be prepared in polyclonal or monoclonal form.
The antibodies of the invention can be raised using S. sobrinus serotype d or streptococcal antigen derived therefrom as the immunogen. Normally, the immunogen will comprise the naturally-occurring streptococcal antigen but, as is the case with the use of streptococcal antigen from serotype c of S. mutans, use can also be made of antigenic fragments of serotype d, provided that those antigenic fragments contain the necessary antigenic determinants characteristic of serotype d.
For the production of polyclonal antibodies, conventional methods may be used involving the immunisation of animals with the serotype d immunogen followed by recovery of the antibodies from the blood of the immunised animals. Conventional antibody recovery methods can be used and conventional antibody purification methods can be used, e.g. affinity chromatography using purified immunogen or fragments thereof.
Where monoclonal antibodies are to be raised, the immunogen derived from serotype d can be used conventionally to immunise mice or other mammals and the spleen of the immunised mammal hybridised with myeloma cells to produce a population of hybridoma cells. Alternatively, immunogen derived from serotype d can be used for in vitro stimulation of B-lymphocytes and the stimulated B-lymphocytes then hybridised with myeloma cells by methods known per se to provide a population of hybridomas. The resulting population of hybridomas may then be screened to select those secreting monoclonal antibody that is cross-reactive by S. sobrinus serotype d and S. mutans serotype c. When polyclonal antibodies are produced, they may also be screened to select those showing the serotype d/c cross-reactivity. The antibodies, however produced, can be purified if necessary by affinity chromatography techniques or by staphylococcal protein A to separate IgG class of antibodies or by using serotype d antigen.
Where antibody fragments are required, the polyclonal or monoclonal antibody produced by the methods described above can be fragmented by digestion with papain or pepsin by conventional methods.